Microorganisms as carriers of nucleotide sequences coding for cell antigens used for the treatment of tumors

ABSTRACT

The invention relates to a microorganism with a nucleotide sequence coding for a cell antigen in which the following components are inserted and are expressible: I) a nucleotide sequence coding for at least one epitope of an antigen of a tumor cell and/or a nucleotide sequence for at least one epitope of an antigen that is specific for a tissue cell from which the tumor originates; II) an optional nucleotide sequence coding for a protein that stimulates cells of the immune system; IIIA) a nucleotide sequence for a transport system which makes it possible to express the expression product of components I) and, optionally, II) on the outer surface of the bacterium and/or secrete the expression product of component I) and, optionally, of component II); and/or IIIB) a nucleotide sequence for a protein used for lysing the microorganisms in the cytosol of mammalian cells and for intracellularly releasing plasmids which are contained in the lysed microorganisms; and IV) an activation sequence for expressing one or several of components I) to IIIB), said activation sequence being selected among the group consisting of an activation sequence which is capable of being activated in the microorganism, is tissue-cell-specific but not cell-specific. Each of components I) to IV) can be identically or differently arranged in an individual or multiple manner. Also disclosed are uses of such a microorganism for the production of a medicament.

FIELD OF THE INVENTION

The invention relates to a microorganism with foreign nucleotidesequences, to the use thereof as a medicament, in particular vaccine, toa plasmid with the foreign nucleotide sequences and a method for theproduction of such a microorganism.

BACKGROUND OF THE INVENTION AND PRIOR ART

The main reason for the in most cases lethal consequence of malignanttumor diseases is the inability of the body's defense system to detectand destroy malignant cancer cells. In the industrial countries, cancerdiseases belong to the most common diseases with lethal course. InGermany alone, more than 210,000 people die per year because ofmalignant new formations (source: WHO, figures of 1997), whichcorresponds to a yearly rate of more than 255 deaths per 100,000inhabitants.

The basis of this invention are newer findings in the molecularmechanisms leading to malignant deformations. In an early stage alreadyof the cancer formation, there are characteristic changes of the controlof cell growth and/or cell differentiation (Pronten, Cancer Surv.32:5-35, 1998). Essentially involved in these changes are proteins ofthe signal transduction and the cell cycle control, which wereidentified in the last years, and all of which are also tumor antigens.

Tumor antigens are roughly divided into three groups (Pardoll, Nat. Med.4:525-531, 1998): i) tumor-specific neoantigens, which exist in thetumor cell in a mutated and/or over-expressed form, such as EGF-R,HER-2, ii) tumor-specific embryonic antigens, such as members of theMAGE protein family or CEA, iii) tumor-tissue-specific differentiationantigens, such as tyrosinase, Mart-1/Melan-A and gp100.

For the effectiveness of a tumor vaccine, an effective induction of CD8+T cells is decisive, since tumor cells do in most cases not representMHC class II molecules, and the intracellularly existing tumor antigensare in most cases MHC class I restringed. For tumor patients, thenaturally occurring populations of CD8+, cytotoxic T cells (CTL), areobviously not sufficient to detect and eliminate the tumor cells(Jaffee, Ann. N.Y. Acad. Sci. 886:67-72, 1999). Furthermore,tumor-specific T cells cannot effectively attack the tumor tissue due tovarious mechanisms (anergy, tolerance, neutralization) (Smyth et al.,Nat Immunol 2:293-299, 2001). A successful vaccine must therefore breakthis anergy or tolerance and induce a sufficient number of activated,specific CTL as well as of specific antibodies. The role of specificantibodies can be seen by the successful use of monoclonal antibodies(mAbs) against tumor antigens of the group (a), such as the alreadycommercially available herceptin, a mAb against HER-2 (Colomer et al.,Cancer Invest 19:49-56, 2001).

It is already known that attenuated intracellular bacteria are suitableas vaccine carriers against certain bacterial infections, which inparticular can be controlled by a so-called Th1 immune response (Hessand Kaufmann, FEMS Immunology & Medical Microbiology 23:165-173, 1999).This response is characterized by CTL and the presence of specific IFN-gsecreting CD4+ T cells (also T helper cells, Th) (Abbas et al., Nature383:787-793, 1996). Other groups have shown that recombinant bacteriacan protect against a heterologous tumor (Medina et al., Eur. J.Immunol. 29:693-699, 1999; Pan et al., Cancer Res. 59:5264-5269, 1999;Woodlock et al., J. Immunother. 22:251-259, 1999; Paglia et al., Blood92:3172-3176, 1998; Paglia et al., Eur. J. Immunol. 27:1570-1575, 1997;Pan et al., Nat. Med. 1:471-477, 1995; Pan et al., Cancer Res.55:4776-4779, 1995). In these cases, however, animals were immunizedagainst a surrogate antigen, and then tumor cells expressing thisantigen were applied.

These tumor systems cannot however be compared to clinical tumors, sincein these models there were no tolerance for the tumor antigen.

A considerable number of different tumor vaccines have already beenclinically investigated. Up to now, however, a break-through for thetreatment of tumor diseases could not be achieved with any of the tumorvaccines or vaccination methods. In view of this background, therecontinues to exist an extremely high need of new tumor therapy methods.

It is known in the art to express expression products of nucleic acidsequences introduced into bacteria on the cell membrane of thesebacteria, or to have them secreted from these bacteria. The basis ofthis technique is the Escherichia coli hemolysin system HlyAsrepresenting the prototype of a type I secretion system of gram-negativebacteria. By means of the HlyAs, secretion vectors were developed, whichpermit an efficient discharge of protein antigens in Salmonellaenterica, Yersinia enterocolitica and Vibrio cholerae. Such secretionvectors contain the cDNA of an arbitrary protein antigen coupled to thenucleotide sequence for the HlyA signal peptide, for the hemolysinsecretion apparatus, hlyB and hlyD and the hly-specific promoter. Bymeans of this secretion vector, a protein can be expressed on thesurface of this bacterium. Such genetically modified bacteria induce asvaccines a considerably higher immune protection than bacteria, in whichthe protein expressed by the introduced nucleic acid remains inside thecell (Donner et al EP 1015023A; Gentschev et al, Gene, 179:133-140,1996; Vaccine 19:2621-2618, 2001; Hess et al PNAS 93:1458-1463, 1996).The disadvantage of this system is however that by the use of thehly-specific promoter, the amount of the protein expressed on theexterior surface of the bacterium is extremely small.

A technique for inserting plasmid DNA into mammalian cells by carrierbacteria such as Salmonella and Listeria monocytogenes was developed.Genes contained in these plasmids could also be expressed in themammalian cells, when they were under the control of a eukaryoticpromoter. Plasmids were introduced into Listeria monocytogenes germs,said plasmids containing a nucleotide sequence for an arbitrary antigenunder the control of an arbitrary eukaryotic promoter. By introductionof the nucleotide sequences for a specific lysis gene, it was obtainedthat the Listeria monocytogenes germs dissolve in the cytosol of theantigen-presenting cell and release their plasmids, which leads to asubsequent expression, processing and presentation of the plasmid-codedproteins and clearly increases the immunogenecity of these proteins(Dietrich et al. Nat. Biotechnol. 16:181-185, 1998; Vaccine19:2506-2512, 2001).

Virulence-attenuated, intracellularly settling bacteria were developed.For instance such variants of Listeria monocytogenes, Salmonellaenterica sv. typhimurium and typhi, and Mycobacterium bovis were alreadyused as well-tolerated live vaccines against typhus and tuberculosis.These bacteria, including their attenuated mutants are generallyimmune-stimulating and can initiate a fair cellular immune response. Forinstance, L. monocytogenes stimulates to a special extent by theactivation of THl-cells the proliferation of cytotoxic T-lymphocytes.These bacteria supply secerned antigens directly into the cytosol ofantigen-presenting cells (APC; macrophages and dendritic cells), whichin turn express the co-stimulating molecules and cause an efficientstimulation of T cells. The listeriae are in part degraded in phagosomalcompartments, and the antigens produced by these carrier bacteria cantherefore on the one hand be presented by MHC class II molecules andthus lead to the induction of T helper cells. On the other hand, thelisteriae replicate after release from the phagosome in the cytosol ofAPCs; antigens produced and secerned by these bacteria are thereforepreferably presented by the MHC class I pathway, thus CTL responsesagainst these antigens being induced. Further it could be shown that bythe interaction of the listeriae with microphages, natural killer cells(NK) and neutrophilic granulocytes, the expression of such cytokines(TNF-alpha, IFN-gamma, IL-2, IL-12; Unanue, Curr. Opin. Immunol.,9:35-43, 1997; Mata and Paterson, J. Immunol. 163:1449-14456, 1999) isinduced, for which an antitumoral efficiency was detected. By theadministration of L. monocytogenes, which were transduced for theexpression of tumor antigens, the growth of experimental tumors could beinhibited antigen-specifically (Pan et al., Nat Med 1:471-477, 1995;Cancer Res. 59:5264-5269, 1999; Voest et al., Natl. Cancer Inst.87:581-586, 1995; Beatty and Paterson, J. Immunol. 165:5502-5508, 2000).

Virulence-attenuated Salmonella enterica strains, into which nucleotidesequences coding for tumor antigens had been introduced, as tumorantigen-expressing bacterial carriers, could provide after oraladministration a specific protection against different experimentaltumors (Medina et al., Eur. J. Immunol. 30:768-777, 2000; Zoller andChrist, J. Immunol. 166:3440-34450, 2001; Xiang et al., PNAS97:5492-5497, 2000).

Recombinant Salmonella strains were also effective as prophylacticvaccines against virus infections (HPV); (Benyacoub et al., Infect Immun67:3674-3679, 1999) and for the therapeutic treatment of a mouse tumorimmortalized by a tumor virus (HPV) (Revaz et al., Virology 279:354-360,2001).

TECHNICAL OBJECT OF THE INVENTION

It is the object of the present invention to provide a medicament, whichin particular represents in the tumor prophylaxis and tumor therapy animproved vaccine for breaking the immune tolerance with respect totumors.

BASIC CONCEPT OF THE INVENTION

For achieving this technical object, the invention teaches amicroorganism with a nucleotide sequence coding for a cell antigen, inthe genome of which the following components are inserted and areexpressible: I) a nucleotide sequence coding for at least one epitope ofan antigen or several antigens of a tumor cell and/or a nucleotidesequence for at least one epitope of an antigen or several antigens thatis or are specific for a tissue cell from which the tumor originates;II) an optional nucleotide sequence coding for a protein that stimulatescells of the immune system; IIIA) a nucleotide sequence for a transportsystem, which makes it possible to express the expression product ofcomponents I) and, optionally, II) on the outer surface of the bacteriumand/or secrete the expression product of component I) and, optionally,of component II); and/or IIIB) a nucleotide sequence for a protein usedfor lysing the microorganisms in the cytosol of mammalian cells and forintracellularly releasing plasmids, which are contained in the lysedmicroorganisms; and IV) an activation sequence for expressing one orseveral of components I) to IIIB), said activation sequence beingselected among the group consisting of “an activation sequence, which iscapable of being activated in the microorganism, istissue-cell-specific, but not cell-specific”, and each of components I)to IV) can be identically or differently arranged in an individual ormultiple manner, and uses of such a microorganism for the production ofa medicament.

Thus, subject matter of the invention are microorganisms, whichrepresent carriers of nucleotide sequences coding for cell antigens,which in turn are expressed or secreted on the outer membrane of themicroorganisms, and the use of these microorganisms for breaking theimmune tolerance against tumors, and new tumor vaccines that containmicroorganisms as carriers of nucleotide sequences coding for cellantigens of normal cells and/or of tumor cells. By the invention, atlast an immune reaction directed against the tumor is caused.

In detail, the microorganisms according to the invention contain thefollowing components: I) at least one nucleotide sequence coding for atleast one epitope of at least one antigen of at least one cell proteinof a tumor cell and/or, optionally, at least one nucleotide sequence forat least one epitope of at least one antigen that is specific for thetissue cell from which the tumor originates; II) optionally, at leastone nucleotide sequence for at least one protein that stimulates cellsof the immune system; IIIA) at least one nucleotide sequence for atransport system for expressing or secreting the cell antigen coded bycomponent I) on the membrane and for secreting the immune-stimulatingprotein coded by component; IIIB) optionally, a nucleotide sequence fora lysine lysing the microorganism in the cytosol, so that plasmids,which are contained in the microorganism, are released into the cytosol;IV) at least one nucleotide sequence for an activation sequence that iscapable to be activated in the microorganism or activated notcell-specifically, but tumor cell-specifically, tissue cell specificallyor function-specifically for expressing components I) and II).

PREFERRED EMBODIMENTS

In the following, the components of a microorganism according to theinvention are described in detail.

Component I).

Component I) represents at least one nucleotide sequence for at leastone epitope of at least one antigen of at least one cell protein or atleast one oncogenically mutated cell protein of a tumor cell. Theoncogenic mutation of the cell protein may have caused a loss or a gainof its original cellular functions. Furthermore, this cell protein canbe selected among the group consisting of “receptor molecules or partsthereof, namely extracellular, transmembranic or intracellular parts ofthe receptors; adhesion molecules or parts thereof, namelyextracellular, transmembranic or intracellular parts of the adhesionmolecules; proteins of the signal transduction; proteins of the cellcycle control; differentiation proteins; embryonic proteins; andvirus-induced proteins”. Such cell antigens perform in the cell thecontrol of the cell growth and of the cell division and are presented onthe cell membrane of normal cells, for instance by the MHC class Imolecule. In tumor cells, these cell antigens are frequentlyover-expressed or specifically mutated. Such mutations can have functionlimitations of oncogene suppressors or the activation of proto-oncogenesto oncogenes as a consequence and can be involved alone or commonly withover-expressions in the tumor growth. Such cell antigens are presentedon the membrane of tumor cells and thus represent antigens on tumorcells, without however causing an immune reaction affecting the tumordisease of the patient. Rapp (U.S. Pat. No. 5,156,841) has alreadydescribed the use of oncoproteins, i.e. expression products of theoncogenes, as an immunogen for tumor vaccines. Reference is explicitlymade to this document.

Examples for cell antigens and their oncogenic mutations according tothe invention are i) receptors, such as Her-2/neu, androgen receptor,estrogen receptor, midkine receptor, EGF receptor, ERBB2, ERBB4, TRAILreceptor, FAS, TNFalpha receptor; ii) signal-transducing proteins andtheir oncogenic mutations, such as c-Raf (Raf-1), A-Raf, B-Raf, Ras,Bcl-2, Bcl-X, Bcl-W, Bfl-1, Brag-1, Mcl-1, A1, Bax, BAD, Bak, Bcl-Xs,Bid, Bik, Hrk, Bcr/abl, Myb, C-Met, IAP1, IAO2, XIAP, ML-IAP LIVIN,survivin, APAF-1; iii) proteins of the cell cycle control and theironcogenic mutations, such as cyclin D(1-3), E, A, B, H, Cdk-1, -2, -4,-6, -7, Cdc25C, P16, p15, p21, p27, p18, pRb, p107, p130, E2F(1-5),GAAD45, MDM2, PCNA, ARF, PTEN, APC, BRCA, P53 and homologues; iv)transcription factors and their oncogenic mutations, such as C-Myc,NFkB, c-Jun, ATF-2, Sp1; v) embryonic proteins, such as carcinoembryonicantigen, alpha-fetoprotein, MAGE, PSCA; vi) differentiation antigens,such as MART, Gp100, tyrosinase, GRP, TCF-4; vii) viral antigens, suchas of the following viruses: HPV, HCV, HPV, EBV, CMV, HSV.

Alternatively or additionally, component I) may represent at least onenucleotide sequence for at least one antigen that is specific for anormal tissue cell, from which the respective tumor originates. Suchspecific antigens are for instance i) receptors, such as androgenreceptors, estrogen receptors, lactoferrin receptors; ii)differentiation antigens, such as basic myelin, alpha-lactalbumin, GFAP,PSA, fibrillary acid protein, tyrosinase, EGR-1, MUC1.

Component II).

Component II) represents at least one nucleotide sequence for at leastone protein, which stimulates cells of the immune system. By theselection of the protein, the immune reaction to the expression productof component I) can be intensified and/or oriented more to theactivation of Th1 cells (for the cellular immune reaction) or to theactivation of Th2 cells (for the humoral immune reaction).Immune-stimulating proteins are for instance i) cytokines, such asM-CSF, GM-CSF, G-CSF; ii) interferons, such as IFN-alpha, beta, gamma;iii) interleukins, such as IL-1, -2, -3, -4, -5, -6, -7, -9, -10, -11,-12, -13, -14, -15, -16, human leukemia inhibitory factor (LIF), iv)chemokines, such as RANTES, monocyte chemotactic and activating factor(MCAF), macrophage inflammatory protein-1 (MIP-1-alpha, beta),neutrophil activating protein-2 (NAP-2), IL-8.

Component IIIA).

Component IIIA) is at least one nucleotide sequence coding for at leastone transport system, which makes it possible to express the expressionof the expression products of components I) and, optionally, II) on theouter surface of the microorganism. The respective component can as anoption be either secreted or expressed on the membrane of themicroorganism, i.e. is membrane-bound. Such transport systems are forinstance i) the hemolysin transport signal of E. coli (nucleotidesequences containing HlyA, HlyB and HlyD under the control of thehly-specific promoter); the following transport signals are to be used:for the secretion—the C-terminal HlyA transport signal, in presence ofHlyB and HlyD proteins; for the membrane-bound expression—the C-terminalHlyA transport signal, in presence of HlyB protein, ii) the hemolysintransport signal of E. coli (nucleotide sequences containing HlyA, HlyBand HlyD under the control of a not hly-specific bacterial promoter),iii) the transport signal for the Slayer protein (Rsa A) of Caulobactercrescentus; the following transport signals are to be used: for thesecretion and the membrane-bound expression—the C-terminal RsaAtransport signal, iv) the transport signal for the TolC proteinEscherichia coli; the following transport signals are to be used: forthe membrane-bound expression—the N-terminal transport signal of TolC(the integral membrane protein TolC of E. coli is a multi-functionalpore-forming protein of the outer membrane of E. coli, which serves—inaddition to functions such as the reception of colicin E1 (Morona etal., J. Bacteriol. 153:693-699, 1983) and the secretion of colicin V(Fath et al., J. Bacteriol. 173:7549-7556, 1991)—also as a receptor forthe U3 phage (Austin et al., J. Bacteriol. 172:5312-5325, 1990); thisprotein is not only found in E. coli, but also in a multitude ofgram-negative bacteria (Wiener, Structure Fold Des 8:R171-175, 2000);the localization in the outer membrane and the wide occurrence make TolCto an ideal candidate to present heterologous antigens, in order e.g. tocause an immune reaction.

Component IIIB).

Component IIIB) is a nucleotide sequence coding for at least one lyticprotein, which is expressed in the cytosol of a mammalian cell and lysesthe microorganism for releasing the plasmids in the cytosol of the hostcell. Such lytic proteins (endolysins) are for instanceListeria-specific lysis proteins, such as PLY551 (Loessner et al MolMicrobiol 16:1231-41, 1995) and/or the Listeria-specific holin under thecontrol of a listerial promoter.

A preferred embodiment of this invention is the combination of differentcomponents IIIB), for instance the combination of a lysis protein andthe holin.

The components IIIA and/or IIIB may be constitutively active.

Component IV).

Component IV) represents at least one nucleotide sequence for at leastone activation sequence for the expression of component I) and,optionally, II).

If the expression is membrane-bound on the outer surface of themicroorganism, the activation sequence has preferably to be selectedsuch that it is capable of being activated in the microorganism. Suchactivation sequences are for instance: i) constitutively active promoterregions, such as the promoter region with “ribosomal binding site” (RBS)of the beta-lactamase gene of E. coli or of the tetA gene (Busby andEbright, Cell 79:743-746, 1994); ii) promoters, which are capable ofbeing induced, preferably promoters, which become active after receptionin the cell. To these belong the actA promoter of L. monocytogenes(Dietrich et al., Nat. Biotechnol. 16:181-185, 1998) or the pagCpromoter of S. typhimurium (Bumann, Infect Immun 69:7493-7500, 2001).

If the plasmids are released from the microorganism after its lysis intothe cytosol of the cell, the activation sequence is not cell-specific,but tissue cell-specific, cell cycle-specific or function-specific.Preferably, such activation sequences are selected, which areparticularly activated in macrophages, dendritic cells and lymphocytes.

Microorganisms in the meaning of the invention are viruses, bacteria orunicellular parasites, which are usually used for the transfer ofnucleotide sequences being foreign for the microorganism.

In a special embodiment of this invention, the microorganisms representgram-positive or gram-negative bacteria, preferably bacteria, such asEscherichia coli, Salmonella, Yersinia enterocolitica, Vibrio cholerae,Listeria monocytogenes, Shigella.

Preferably, such bacteria are used, which are attenuated in theirvirulence.

The components according to the invention are introduced into themicroorganisms by methods well known to the man skilled in the art. Ifthe microorganisms represent bacteria, the components are inserted intoplasmids, and the plasmids are transferred into the bacteria. Thetechniques suitable for this and the plasmids are sufficiently known tothe man skilled in the art.

Subject matter of the invention are medicament preparations containingthe microorganisms according to the invention or however membraneenvelopes of these microorganisms. The preparation of these membraneenvelopes takes for instance place according to the method described inEP-A-0,540 525. Such medicament preparations are for instancesuspensions of the microorganisms according to the invention in thesolutions familiar to the pharmacist, suitable for injection.

Another subject matter of the invention is the administration of amedicament preparation containing the microorganisms according to theinvention. The administration is made locally or systemically, forinstance into the epidermis, into the subcutis, into the musculature,into a body cavity, into an organ, into the tumor or into the bloodcirculation.

A particular subject matter of this invention is the peroral or rectaladministration of the medicament according to the invention for theprophylaxis and/or therapy of a proliferative disease. Theadministration can be made once or several times. In eachadministration, approximately 10 to 10ˆ9 microorganisms according to theinvention are administered. If the administration of this number ofmicroorganisms according to the invention does not cause a sufficientimmune reaction, the number to be injected has to be increased.

After administration of the microorganisms according to the invention,the tolerance for a cell presenting component I), for instance for atumor cell, or for a tissue cell, from which the tumor originates, isbroken, and a cytotoxic immune reaction directed against the tumorand/or its tissue cells is triggered.

Depending on the selection of component I), this cytotoxic immunereaction is directed either exclusively against the tumor or alsoagainst the tumor cells including the tissue cells, from which the tumorcells originate.

Subject matter of the invention is thus the administration of amedicament preparation according to the invention for the prophylaxis ortherapy of a proliferative disease. Proliferative diseases are tumordiseases, leukemias, virally caused diseases, chronic inflammations,rejections of transplanted organs and autoimmune diseases.

In a special embodiment of this invention, wherein component I)represents at least one cell antigen, which is expressed by a tumor celland the tissue cells, from which the tumor originates, the medicamentaccording to the invention is administered for the prophylaxis ortherapy of a tumor of the glandula thyroidea, the mamma, the stomach,the kidney, the ovarium, the nevi, the prostate, the cervix or thevesica urinaria.

In the following, the invention is explained in more detail, based onexamples representing embodiments only.

EXAMPLE 1 Induction of an Immune Response in BxB Mice by Immunizationwith Salmonellae Expressing c-raf

Raf is a normally cytosolic serine/threonine kinase (PSK), which inconjunction with other proteins of signal cascades controls the cellgrowth and survival (Kerkhoff and Rapp, Oncogene 17:1457-1462, 1998;Troppmair and Rapp, Recent Results Cancer Res. 143:245-249, 1997). Abinding of a growth factor to a respective receptor normally leads viaan activation of Ras, the subsequent activation of Raf via severalphosphorylation steps via the PSK and tyrosine kinase MEK and the PSKERK to an activation of the replication machinery in the cell nucleus(Kerkhoff and Rapp, Oncogene 17:1457-1462, 1998). The first link in thischain, the small G protein Ras, is present in a modified form in 30% ofall human tumors (Zachos and Spandidos, Crit. Rev. Oncol. Hematol.26:65-75, 1997). Raf is an effector of Ras and is present in anover-expressed form in a multitude of human tumors (Naumann et al.,Recent Results Cancer Res. 143:237-244, 1997).

For the test in the mouse model, transgenic mice were used, whichover-express the complete molecule or the constitutively active kinasedomain (BxB) (Kerkhoff et al., Cell Growth Differ 11:185-190, 2000).Therewith, the mice spontaneously develop lung tumors approx. half ayear later.

For the generation of the vaccines, the human c-Raf cDNA was cloned bymeans of PCR in-frame with HlyA into the plasmid pMOhly 1 (FIG. 1).Subsequently, the plasmid pMO-Raf was transfected into attenuatedsalmonellae (S. typhi murium SL7207), which carry a defect in thearomatic metabolism (Hoiseth and Stocker, Nature 291:238-239, 1981). Inthe immune blotting by means of antibodies directed against c-Raf, thec-Raf HlyAs fusion protein could be detected in the bacterium lysate aswell as in the culture supernatant of SL7207 bacteria transfected withPMOhy-Raf.

BxB transgenic mice were orally immunized at an age of 7-10 weeks withthe salmonellae (dose 5×10ˆ9), and the vaccination was repeated twice inan interval of 5 days. 45 days after the last immunization, anintravenous refreshing vaccination with 5×10ˆ5 salmonellae was made. Forcontrol purposes, naked c-Raf coding DNA was intramuscularlyadministered to the mice.

5-7 days after the last immunization, now serum samples were taken, andthe antibody response was analyzed by means of a Western blot. For thispurpose, the 1:200 diluted serum was hybridized against membranes withseparated protein and blotted protein of c-Raf-transfected or nottransfected bacteria. The detection of the bound serum antibodies tookplace by means of antibodies specific for mouse IgG. In contrast to thecontrol mice, immunized with pMohly-Raf transfected SL7207,c-Raf-specific antibodies of the isotype IgG could be induced. Thus ithas been shown that an immunization with the described salmonellae canbreak the self-tolerance and induces CD4+ T cells, which are necessaryfor the antibody isotype change to IgG.

For the analysis of the CD8+ T cell response, C57BL-6 mice wereimmunized following the same protocol. 7 days after the lastimmunization, spleen cells were isolated, and they were stimulated withRaf-over-expressing EL-4 cells. 1 h after beginning the stimulation, thevesicular transport was blocked by Brefeldin A, and after another 4 h,the cells were stained with CD8 and IFN-g-specific antibodies andanalyzed by flow cytometry (Mittrucker et al., Infect Immun 70:199-203,2002). Only in one pMO-Raf-immunized mouse, a Raf-specific antibodyresponse could be detected.

For detecting the tumoricidal activity, 10, 12 and 14 months oldimmunized and not immunized BxB mice were killed, and the lung mass wasweighed. The lung mass is a direct measure for the size of the tumor. Inthe group, immunized with SL-pMO-Raf, after 14 months clearly morefrequently mice with a reduced lung mass could be found than in thecontrol groups including the group, which has been immunized with nakedDNA coding for c-Raf (SL-pCMV-raf). Normally, the tumor growth on nottreated animals is not reversible (Kerkhoff et al., Cell Growth Differ.11:185-190, 2000). These data thus show that in this experiment avaccination with SL-pMO-Raf animals could protect from the generation oftumors, and the invention described here is suitable as a tumor vaccine.

These experiments further show that the carrier system represented inthis invention can in principle break the self-tolerance and induce inc-Raf-tolerant animals a c-Raf-specific antibody response and T cellresponse.

By means of the same experimental systems, salmonellae can be producedas vaccines, which express isoforms of C-Raf (such as for instance B-Rafand A-Raf), mutated C-Raf, B-Raf or A-Raf, epitopes of normal or mutatedC-Raf, B-Raf or A Raf, or combinations of epitopes of normal and/ormutated C-Raf, B-Raf or A-Raf. Examples for a mutation coming along witha loss of the activity of Raf are mutations of the Ras-binding domain,the kinase domain and/or the phosphorylation sites.

EXAMPLE 2 Induction of an Immune Response in BALB/C Mice by Immunizationwith Salmonellae Expressing PSA

The existence of tissue-specific antigens, in particular of those, whichare synthesized and expressed to a high degree by tumor cells, is,beside the diagnostic usability of these markers, also a possiblestarting point for therapeutic approaches. For the prostate carcinoma,up to now three antigens worth mentioning have been identified: PSA(prostate-specific antigen), PSMA (prostate-specific membrane antigen)and PSCA (prostate stem cell antigen). Whilst PSA exists already inearly tumor forms in an over-expressed manner (Watt et al., Proc. Natl.Acad. Sci. USA 83:3166-3170, 1986; Wang et al., Prostate 2:89-96, 1981)and thus contributes for carcinoma diagnosis (Labrie et al., J. Urol.147:846-851; discussion 851-842, 1992), the PSCA expression is in mostcases only increased in the locally advanced, dedifferentiated andmetastasized tumor stage (Gu et al., Oncogene 19:1288-1296, 2000; Reiteret al., Proc. Natl. Acad. Sci. USA 95:1735-1740, 1998). The organspecificity makes PSA as well as PSCA to a potential target antigen forthe development of immune therapies against the prostate carcinoma(Reiter et al., Proc. Natl. Acad. Sci. USA 95:1735-1740, 1998; Hodge etal., Int. J. Cancer 63: 231-237, 1995; Armbruster, Clin. Chem.39:181-195, 1993).

In this first experiment, it was intended to show whether PSA-secerningsalmonellae on the base of the vector pMOHLY 1 can induce an immuneresponse in BALB/c mice. For this purpose, first two NsiI interfaceswere introduced by polymerase chain reaction (PCR) into the c-DNAsequence of PSA, in order to make an in-frame insertion of the amplifiedfragment into the target vector possible. For the amplification, afragment of 645 base pairs (bp) was selected. As primers served5′-GTGGATTGGTGATGCATCCCTCATC-3′ and 5′-CAGGGCACATGCATCACTGCCCCA-3′. ThePCR product was first cloned blunt-end into the vector pUC18 and laterligated via NsiI interfaces with the target vector pMOhlyl. The correctinsertion was controlled by means of restriction digestion and confirmedby sequentiation (FIG. 2).

By means of this salmonella strain, BALB/c mice were now nasallyimmunized three times in an interval of 3 weeks with a dose of 1×10⁷.The immune response is detected with Western blot analyses andintracellular cytokine staining.

1. A microorganism with a nucleotide sequence coding for a cell antigen,in the genome of which the following components are inserted and areexpressible: I) a nucleotide sequence coding for at least one epitope ofan antigen or several antigens of a tumor cell or a nucleotide sequencefor at least one epitope of an antigen or several antigens that is orare specific for a tissue cell from which the tumor originates; II) anucleotide sequence coding for a protein that stimulates cells of theimmune system; IIIA) a nucleotide sequence for a transport system, whichmakes it possible to express the expression product of components I)IIIB) a nucleotide sequence for a protein for lysing the microorganismsin the cytosol of mammalian cells and for intracellularly releasingplasmids, which are contained in the lysed microorganisms; and IV) anactivation sequence for expressing one or several of components 1) toIIIB), said activation sequence being selected from the group consistingof an activation sequence, which is capable of being activated in themicroorganism, or which is tissue-cell-specific, or which is notcell-specific, wherein each of components 1) to IV) can be identical ordifferent, and each present once or multiple.
 2. The microorganismaccording to claim 1, wherein the microorganism is a virus or abacterium comprising a gram-positive or gram-negative bacterium, furthercomprising Escherichia coli, Salmonella, Yersinia enterocolitica, Vibriocholerae, Listeria monocytogenes, and Shigella, or is a unicellularparasite, the virulence of the microorganism being reduced.
 3. Themicroorganism according to claim 1, wherein the microorganism is theenvelope of a bacterium.
 4. The microorganism according to claim 1,wherein component I) is a nucleotide sequence coding for an epitope orseveral epitopes of an antigen or several antigens of a protein orseveral proteins of a tumor cell, wherein this protein comprisesextracellular, transmembranic or intracellular part of a receptor;extracellular, transmembranic or intracellular part of an adhesionmolecule; signal-transducing protein; a protein controlling the cellcycle; transcription factor; differentiation protein; embryonic protein;and viral protein, wherein the protein is an oncogenic gene product or asuppressor gene product comprising c-raf, A-Raf, B-Raf or a homologousprotein of c-Raf, A-Raf or B-Raf.
 5. The microorganism according toclaim 1, wherein component I) is a nucleotide sequence coding for anantigen that is specific for the tissue cell comprising glandulathyroidea, glandula mammaria, glandula salivaria, nodus lymphoideus,glandula mammaria, tunica mucosa gastris, kidney, ovarium, prostate,cervix, tunica serosa vesicae urinariae and nevus, from which the tumororiginates.
 6. The microorganism according to claim 1, comprising acomponent I) according to claim 4 and a component I) according to claim5.
 7. The microorganism according to claim 1, wherein component II)codes for at least one cytokine, interleukin, interferon or chemokine.8. The microorganism according to claim 1, wherein component IIIA) codesfor the hemolysin transport signal of Escherichia coli, the Slayer (RsaA) protein of Caulobacter crescentus or for the TolC protein ofEscherichia coli.
 9. The microorganism according to claim 1, whereincomponent IB) codes for a lytic protein of gram-positive bacteria, alytic protein of Listeria monocytogenes, for PLY551 of Listeriamonocytogenes or the holin of Listeria monocytogenes.
 10. Themicroorganism according to claim 1, wherein component IV) codes for anactivator sequence capable of being activated in the microorganismcomprising a tumor cell-specific, tissue cell-specific,macrophagespecific, dendrite-specific, lymphocyte-specific,function-specific activator sequence or an activator sequence beingcell-non-specifically activated.
 11. The microorganism according toclaim 1, wherein component I) codes for at least two different proteins.12. A method for the prophylaxis or therapy of a disease, which iscaused by uncontrolled cell division or an infection comprising a tumordisease, further comprising a prostate carcinoma, an ovary carcinoma, amamma carcinoma, a stomach carcinoma, a kidney tumor, a tumor ofglandula thyroidea, a melanoma, a tumor of cervix, a tumor of vesicaurinaria, a tumor of glandula salivaria or a tumor of nodus lymphoideus,a leukemia, a viral or bacterial infection, a chronic inflammation, anorgan rejection or an autoimmune disease comprising administering aphysiologically effective dose of a medicament comprising amicroorganism according to claim
 1. 13. The method according to claim 12further comprising the removal of a tumor as well as of the healthytissue from which the tumor originates.
 14. The method according toclaim 12, wherein the medicament is prepared for local, parenteral, oralor rectal administration.
 15. A method for the production of amedicament according to claim 12, wherein a microorganism according toclaim 1 is prepared in a physiologically effective dose with one orseveral physiologically tolerated carrier substances for oral,intramuscular, intravenous, intraperitoneal, rectal or localadministration.
 16. A plasmid or expression vector comprising thecomponents I) to IV) according to claim
 1. 17. A method for theproduction of a microorganism according to claim 1, wherein a plasmid orexpression vector according to claim 16 is produced, and a microorganismis transformed with this plasmid or expression vector.
 18. Themicroorganism of claim 1, wherein the nucleotide sequence of componentIIIA) is capable of causing the expression of the expression product ofcomponent I) on the outer surface of the bacterium, or secretion of theexpression product of component I).
 19. The microorganism of claim 1,wherein the nucleotide sequence of component IIIA) is capable of causingthe expression of the expression product of component II).